Biomedical scientists require ever improving tools to face the complex challenge of understanding the intricacies of protein regulation and its relationship to disease states. Recent research in the field of protein biochemistry has revealed that in many cases oxidative chemical modifications to protein molecules actually serve as a means of regulating protein activity helping to absorb, informationally register (for the purpose of activating biological feedback mechanisms) and/or alter protein activity, and deflect what otherwise might have been injurious insults. Cysteine sulfenic acid formation stands as one such particularly difficult-to-track modification (due to its inherent instability outside of its native cocoon-like protein environment) that appears to play a key role in the biochemical regulation of many proteins. The analytically elusive nature of this protein modification has made understanding its biological role and thus the full biological role of any affected protein an oft-ignored priority. The driving hypothesis of this application is that oxidative [redox] regulation of proteins via cysteine sulfenic acid formation critically affects the activity of proteins containing one or more unoxidized cysteine residues [i.e., cysteine residues with free thiol functional groups]. The long term goal of this research is, thus, to develop methodology that facilitates the amino acid residue-specific identification and quantitation of cysteine sulfenic acid modifications of specific target proteins directly out of their biological environment such as might be appropriate for making clinical diagnoses. This goal will be realized through completion of three specific aims: 1) Synthesize a custom-designed trifunctional molecular tag that can be used to specifically label site(s) of cysteine sulfenic acid within proteins and simultaneously provide a handle for affinity purification and a signature pattern in preliminary analyses [within single stage mass spectra] that indicates the presence and molecular mass of labeled peptides. 2) Optimize the preparation of blood plasma samples for qualitative identification and relative quantitation of cysteine sulfenation events within specifically targeted proteins. And 3) validate preparatory and analytical procedures according to the Association of Analytical Chemists-recommended method validation criteria and begin to qualitatively and quantitatively test biologically extracted, genuinely unknown, normatively unoxidized [free thiol-containing] proteins for cysteine sulfenic acid modifications. Recent evidence shows that within proteins, a uniquely modified (oxidized) form of the amino acid cysteine known as cysteine sulfenic acid can play important regulatory roles in both day-to-day biological function and in disease states. Because of their unstable nature, however, the biologically important details of such modifications go all but unnoticed under traditional techniques used to directly analyze proteins. The project under proposal here aims to remedy this analytical deficit by providing a robust means to study the detailed qualitative and quantitative nature of cysteine sulfenic acid modifications in proteins extracted directly from clinically relevant biological samples.